Kaempferol Addition Increases the Antimalarial Activity of Artesunate in Experimental Mice

Kaempferol (KMF) is a member of flavonol widely found in tea, broccoli, apples, strawberries, and beans. It has been demonstrated to present several pharmacological properties with potent antimalarial activity against Plasmodium berghei-infected mice. Hence, the search for a safe and new antimalarial compound with combinations to delay the development of resistance was the aim of this study. Thus, the therapeutic effect of the combination of KMF and artesunate (ART) in P. berghei-infected mice was evaluated. Combination of KMF and ART in P. berghei ANKA- (PbANKA-) infected ICR mice in a fixed-ratio combination (1 : 1) and fractions of their median effective dose (ED50) was also investigated using the standard 4-day suppressive test. The ED50 levels of KMF and ART in mice infected with PbANKA were 20.06 ± 2.65 and 6.06 ± 1.33 mg/kg, respectively. Moreover, KMF showed promising synergistic combination with ART at the doses of their ED50 and fixed-ratio combination (1 : 1) of their ED50 of 1/2 with combination index (CI) values of 0.86 and 0.47, respectively. Additionally, KMF, ART, and its combination at the doses of their ED50 and fixed-ratio combination (1 : 1) of their ED50 of 1/2 also presented significantly (P < 0.001) prolonged mean survival time (MST). The findings of this study showed that a combination of KMF and ART enhanced the antimalarial activity of ART and prolonged MST. This study supports the basis for the selection of KMF as a prospective compound for further consideration as a partner drug for ART.


Introduction
Malaria remains one of the leading causes of mortality and morbidity accounting for about 10% of childhood deaths in sub-Saharan Africa [1]. In 2018, it was estimated that 228 million cases of malaria and 405,000 malaria-related deaths occurred in the world [2]. e resistance of Plasmodium parasites to available antimalarial drugs especially to artemisinin and the delayed parasite clearance with the artemisinin-based combination therapy (ACT) has led to increase treatment failure [3][4][5][6]. Hence, the search for safe and selective chemotherapeutic drugs with efficacy that will not be compromised by Plasmodium remains the focus.
Kaempferol, a flavonol widely found in different vegetables (tea, broccoli, apples, strawberries, and beans), is known to be one of the most active and important natural compounds with several pharmacological properties including antioxidant, anti-inflammatory, antimicrobial, and anticancer [7][8][9]. Moreover, kaempferol has been studied and found to be an important compound in the search for an effective antimalarial drug against Plasmodium falciparum and Plasmodium berghei-infected mice [10,11]. However, the possible application of a combination of kaempferol with widely used artemisinin derivative, artesunate, in combating malaria has not yet been investigated. erefore, this study was aimed to investigate the therapeutic effect of a combination of kaempferol and artesunate in P. berghei-infected mice using in vivo model.

Drugs and Reagents.
Artesunate (ART), kaempferol (KMF), and Tween-80 were obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents were of analytical grade. e selected doses of ART and KMF were prepared in 20% Tween-80 and administered orally by gavage. e untreated group was given 20% Tween-80 only.

Experimental
Mice. ICR male mice, 4-6 weeks old, weighing 25-30 g at the time of primary infection were used throughout this study. ey were obtained from the National Laboratory Animal Center, Mahidol University, ailand, and kept in an animal room with temperatures of 22-25°C, 12 h light-dark cycle. e animals were allowed free access ad libitum to a pellet diet and clean water. All experiments involving experimental mice were approved by the Animal Ethical Committee of Walailak University (AE002/2019).

Rodent Malaria Parasite.
In this study, chloroquinesensitive Plasmodium berghei strain ANKA (PbANKA) obtained from Malaria Research and Reference Reagent Resource Center (MR4, https://www.beiresources.org/ About/MR4.aspx) was used. e parasite was maintained by intraperitoneal (IP) injection of 1 × 10 7 parasitized erythrocytes of PbANKA into naïve ICR mice. Propagation of parasite was monitored by microscopy of Wright-Giemsa stained blood film, and percent parasitemia was subsequently calculated using % parasitemia � number of parasitized erythrocytes number of total erythrocytes × 100. (1)

Antimalarial Suppressive
Test. e ED 50 of ART and KMF was estimated using the standard 4-day suppressive test as previously described [12]. Forty-five naïve ICR mice were infected with 1 × 10 7 parasitized erythrocytes of PbANKA by IP injection. After 2 h, the mice were divided into 9 groups of 5 mice each. e test groups were given KMF (1, 5, 20, and 100 mg/kg) and ART (1, 3, 10, and 30 mg/kg) orally. e untreated group received 10 ml/kg of 20% Tween-80. e drugs were treated once a day for 4-consecutive days (D0-D3). At D4, parasitemia was estimated using microscopy of Wright-Giemsa stained thin blood film, and percent parasite suppression was subsequently calculated using % inhibition � (parasitemia of untreated group − parasitemia of treated group) parasitemia of untreated group × 100. (2)

In Vivo Antimalarial Combination Test.
In order to obtain the combination potency of the ART and KMF, antimalarial activity was assayed using the standard 4-days suppressive test previously described above. Twenty naïve ICR mice were administered by IP infection with 1 × 10 7 parasitized erythrocytes of PbANKA. After 2 h, the mice were divided into 5 groups of 5 mice each and given the combination drugs at the doses of their respective ED 50 and fixed-ratio combination (1 : 1) of their respective ED 50 1/2, 1/4, and 1/8. e untreated group was given 10 ml/kg of 20% Tween-80. e treatment was carried out once a day for 4-consecutive days (D0-D3). Parasitemia and percent suppression of parasite growth were subsequently calculated on D4.

Determination of Mean Survival Time.
e mortality of all experimental mice was daily monitored, and the number of days from parasite inoculation up to death was recorded throughout the follow-up period. e mean survival time (MST) was calculated using the following formula [13]: MST � sum of survival time of all mice in a group total number of mice in that group .
2.7. Statistics. All results were expressed as the mean-± standard error of the mean (SEM) using GraphPad Prism (GraphPad Software version 5.01, Inc., USA). e nonlinear regression function for the sigmoidal dose-response variable slope was used to calculate the best-fit ED 50 . One-way ANOVA followed by Tukey's post hoc test was used to compare the mean of measured parameters. e P < 0.05 at 95% confidence was regarded as statistically significant. Moreover, a combination index (CI), which defines synergism (CI < 1), additive effect (CI � 1), and antagonism (CI > 1), was automatically simulated by CompuSyn (CompuSyn software, ComboSyn, Inc., USA) [14].

Antimalarial Activity of KMF and ARTagainst PbANKA-Infected
Mice. e chemosuppressive antimalarial activity of KMF and ART against PbANKA-infected mice is shown in Figure 1. KMF presented a significant (P < 0.05) dose-dependent inhibition in parasitemia levels with similar inhibition as in ART-treated groups. From the dose-response curve, the ED 50 of KMF and ART was 20.06 ± 2.65 and 6.06 ± 1.33 mg/kg, respectively.  Journal of Tropical Medicine

Combination of KMF and ARTagainst PbANKA-Infected
Mice. e combination of KMF and ART at the doses of their respective ED 50 and fixed-ratio combination (1 : 1) of their respective ED 50 of 1/2 exerted a significant (P < 0.001) reduction of parasitemia, compared to the untreated group with the CI values of 0.86 and 0.47, respectively, indicating synergism ( Figure 2 and Table 1). Additionally, significant (P < 0.05) inhibition was also observed in these combinations when compared with 6 mg/kg of the ART-treated group. However, the combination of KMF and ART at the doses of their respective at 1/4 ED 50 and 1/8 ED 50 did not show the significant inhibition as compared to untreated and 6 mg/kg of ART-treated groups with the CI values of 4.64041 and 85.9454, respectively, indicating antagonism (Table 1).

Discussion
ACT is now the major strategy for malarial treatment due to the high efficacy and low probability of antimalarial drug resistance development [15]. is study focused on the combination of KMF and ART, with the aim of developing a new ACT. Using the chronic toxicity test, KMF in mice has been previously reported to be safe at the dose up to 2,000 mg/kg in mice [10]. Moreover, the susceptibility test in the suppressive model of KMF against PbANKA showed potent antimalarial activity in a dosedependent manner. Accordingly, this was a basis for the selection of the doses (1, 5, 10, 20, and 100 mg/kg) of KMF for the present ED 50 evaluation. KMF exerted a significant (P < 0.05) dose-dependent antimalarial activity with similar inhibition as in ART-treated groups. It was due to the fact that antioxidant and anticancer activities of KMF might be responsible for its antimalarial activity [7,10,11]. For our finding, the present study reports for the first time ED 50 level of KMF in the antimalarial in vivo model. e combination treatment (1 : 1) of ED 50 values of KMF and ART produced a more significant inhibition in parasitemia compared to the use of only ART. e synergistic effect was pronounced when the KMF and ART were used   together at the doses of 1/2 ED 50 each. is may be related to a rapid onset of antimalarial activity of KMF in combination with ART. Unfortunately, the mode of action involved in the enhanced antimalarial activity obtained with KMF and ART combinations has not yet been investigated. Further studies should be performed to highlight the mechanisms behind its antimalarial interactions. Additionally, the combination of KMF and ART at the doses of ED 50 1/4 and ED 50 1/8 displayed antagonistic effect. is could be due to the subtherapeutic doses used.
It has been described that the tested compound that could significantly prolong the MST of infected mice is considered as active [13]. For the result of MST obtained from this study, PbANKA-infected mice treated with KMF, ART, and its combination had significantly longer MST than the untreated group. is might be due to the antimalarial activity of these compounds.

Conclusion
From this study, it can be concluded that the combination at a fixed ratio (1 :1) of KMF and ART exerted potent antimalarial activity with a synergistic effect against PbANKA-infected mice. In addition, significant prolonged MST in PbANKAinfected mice treated with these drugs was also observed. Accordingly, KMF could serve as a potential partner drug that can be combined with ART for the treatment of malarial infection.

Conflicts of Interest
e authors have declared that there are no conflicts of interest regarding the publication of this manuscript.